Method for treating cancers

ABSTRACT

The invention provides for the production of several humanized murine antibodies specific for the antigen LK26, which is recognized by the murine antibody LK26. This antigen is expressed on all choriocarcinoma, teratocarcinoma and renal cancer cell lines whereas it is not expressed on cell lines of leukaemias, lymphomas, neuroectodermally-derived and epithelial tumor cell lines (excepting a small subset of epithelial cell lines). Furthermore, whereas renal cancer cell lines express the LK26 antigen, normal renal epithelial cells do not. Similarly, with the exception of the trophoblast, all normal adult and fetal tissues tested are negative for the LK26 phenotype. The invention also provides for numerous polynucleotide encoding humanized LK26 specific antibodies, expression vectors for producing humanized LK26 specific antibodies, and host cells for the recombinant production of the humanized antibodies. The invention also provides methods for detecting cancerous cells (in vitro and in vivo) using humanized LK26 specific antibodies. Additionally, the invention provides methods of treating cancer using LK26 specific antibodies.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/266,119,filed Mar. 10, 1999 now U.S. Pat. No. 6,124,106 which is a divisional ofSer. No. 08/760,840 filed Dec. 5, 1996, now U.S. Pat. No. 5,952,484which is a divisional of Ser. No. 08/207,996, filed Mar. 8, 1994, nowU.S. Pat. No. 5,646,253.

1. FIELD OF THE INVENTION

The present invention is related to the field of molecular biology, andmore particularly to humanized antibodies.

2. BACKGROUND

The present invention provides novel recombinant immunoglobulinsspecific for the human LK26 cancer antigen, polynucleotides encoding thenovel immunoglobulins, and host cells containing the novelpolynucleotides. The invention also provides methods for the productionof these recombinant antibodies, for the diagnosis and treatment ofcertain human cancers.

Transformation of a normal cell to a malignant cell is often accompaniedby a change in the expression of cell surface antigens. These changes inthe cell surface can be detected using monoclonal antibodies specificfor such antigens. In this way, different cancer cells can be detectedand characterized (Lloyd, K. O. (1983) “Human Tumour Antigens: Detectionand Characterization with Monoclonal Antibodies” in R. B. Herberman,ed., Basic and Clinical Tumour Immunology, pp 159-214, Martinus Nijhoff,Boston).

European Patent Application Number 86104170.5 (Rettig) describes thegeneration and characterization of the murine monoclonal antibody‘LK26’. The antibody was generated by the application of the hybridomatechnology of Kohler and Milstein (Kohler, G. and Milstein, C. (1975)Nature 256:495-497). The antibody specifically recognizes a cell surfaceglycoprotein of molecular weight between 30 and 35 kDa (the LK26antigen). This antigen is expressed on all choriocarcinoma,teratocarcinoma and renal cancer cell lines whereas it is not expressedon cell lines of leukaemias, lymphomas, neuroectodermally-derived andepithelial tumour cell lines (excepting a small subset of epithelialcell lines). Furthermore, whereas renal cancer cell lines express theLK26 antigen, normal renal epithelial cells do not. Similarly, with theexception of the trophoblast, all normal adult and fetal tissues testedare negative for the LK26 phenotype.

The specificity of the LK26 murine antibody makes it a powerful tool forthe detection and characterization of particular human cancer types invitro. However, the in vivo use of murine antibodies as agents for thediagnosis and treatment of human diseases is severely curtailed by anumber of factors. Specifically, the human body recognizes murineantibodies as foreign. This can elicit a human anti-mouse antibody(HAMA) response (Schroff, R. et al. (1985) Cancer Res. 45:879-885) whichresults in rapid clearance of the antibody from the circulation.Furthermore, the Fc portion of a murine antibody is not as efficaciousas the human Fc at stimulating human complement or cell-mediatedcytotoxicity. Therefore, it is desirable to circumvent these problemsassociated with the in vivo use of murine antibodies in diagnosis andtherapy.

EP120694 (Celltech) and EP125023 (Genentech) disclose the development of‘chimeric’ antibodies using recombinant DNA methods. Such antibodiescomprise the variable regions from one species, e.g. mouse, and theconstant regions from another species, e.g. human. Such chimericantibodies have the advantage that they retain the specificity of themurine antibody but can also stimulate human Fc dependent complementfixation and cell-mediated cytotoxicity. However, the murine variableregions can still elicit a HAMA response (Bruggemann, M. et al. (1989)J. Exp. Med. 170:2153-2157) thereby limiting the value of chimericantibodies as diagnostic and therapeutic agents.

British Patent Application Number GB2188638A (Winter) discloses aprocess whereby recombinant antibodies can be generated by substitutionof only the variable region CDRs of one antibody with those fromanother. Typically, this ‘CDR-grafting’ technology has been applied tothe generation of recombinant, pharmaceutical antibodies consisting ofmurine CDRs, human variable region frameworks and human constant regions(e.g. Riechmann, L. et al., (1988) Nature 332:323-327). Such ‘reshaped’or ‘humanized’ antibodies have less murine content than chimericantibodies and retain the human constant regions necessary for thestimulation of human Fc dependent effector functions. Consequently,humanized antibodies are less likely than chimeric antibodies to evoke aHAMA response when administered to humans, their half-life incirculation should approach that of natural human antibodies and theirdiagnostic and therapeutic value is enhanced.

In practice, simply substituting murine CDRs for human CDRs is notsufficient to generate efficacious humanized antibodies retaining thespecificity of the original murine antibody. There is an additionalrequirement for the inclusion of a small number of critical murineantibody residues in the human variable region. The identity of theseresidues depends on the structure of both the original murine antibodyand the acceptor human antibody. British Patent Application Number9019812.8 describes a method for identifying a minimal number ofsubstitutions of foreign residues sufficient to promote efficaciousantigen binding.

The invention described herein provides a process for the inclusion ofresidues into the humanized antibodies which facilitates the properassociation of the VH and VL domains and thereby antigen binding.

The present invention provides novel, humanized monoclonal antibodiesspecific for the human LK26 cancer antigen and various antibodyderivatives comprising humanized variable regions that are specific forLK26. This has been achieved by the conversion of the murine LK26monoclonal antibody described in European Patent Application Number86104170.5 to humanized antibodies by utilizing CDR-graftingtechnologies. The invention also provides methods for the production ofthese humanized antibodies to be used in the diagnosis (both in vivo andin vitro) and treatment of certain human cancers. Prior to the work ofthe inventors, it was not known that the LK26 antibody or any othernon-human antibody specific for the the LK26 antigen could be humanizedso as to retain useful binding specificity.

3. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the DNA sequence (SEQ ID NO: 26 and SEQ ID NO: 27) andcorresponding amino acid sequence (SEQ ID NO: 28) of the murine LK26heavy chain variable region (VH). The CDRs are boxed. Underlinednucleotides and amino acid residues are derived from the oligonucleotideprimers used. The backslash mark is used to indiacte the result obtainedwith the consensus primers.

FIG. 2 shows the DNA sequence (SEQ ID NO: 29 and SEQ ID NO: 31) andcorresponding amino acid sequence (SEQ ID NO: 30) of the murine LK26light chain variable region (VK). The CDRs are boxed. Underlinednucleotides and amino acid residues are derived from the oligonucleotideprimers used.

FIG. 3 shows the vector pSVgpt, which is used for the expression ofchimeric or humanized heavy chains in mammalian cells.

FIG. 4 shows the vector pSVhyg for the expression of chimeric orhumanized light chains in mammalian cells.

FIGS. 5-12 provide graphical data of ELISA results measuring the bindingof different humanized LK26 antibodies.

4. SUMMARY OF THE INVENTION

One aspect of the invention is to provide humanized antibodies specificfor the LK26 antigen.

Another aspect of the invention is to provide polynucleotides encodinghumanized antibodies specific for the LK26 antigens. Various expressionvectors comprising polynucleotides encoding humanized LK26 antibodiesjoined to promoter sequences are also provided. Similarly, anotheraspect of the invention is host cells transformed with expressionvectors for the expression of humanized LK26 specific antibodies.

Another aspect of the invention is to provide humanized anti-LK26antibodies that are labeled with a detectable label or a therapeuticlabel.

Another aspect of the invention is to provide methods for treatingand/or diagnosing cancer by administering a composition comprising ahumanized LK26 specific antibody. One method of detecting cancer cellsinvolves the steps of administering a labeled antibody (detectablelabel) to a patient and subsequently detecting where in the body thelabeled antibody has bound.

5. DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

As used herein, the term “humanized” antibody refers to a molecule thathas its CDRs (complementarily determining regions) derived from anon-human species immunoglobulin and the remainder of the antibodymolecule derived mainly from a human immunoglobulin. The term “antibody”as used herein, unless indicated otherwise, is used broadly to refer toboth antibody molecules and a variety of antibody derived molecules.Such antibody derived molecules comprise at least one variable region(either a heavy chain of light chain variable region) and includemolecules such as Fab fragments, Fab′ fragments, F(ab′)₂ fragments, Fdfragments, Fabc fragments, Fd fragments, Fabc fragments, Sc antibodies(single chain antibodies), diabodies, individual antibody light chains,individual antibody heavy chains, chimeric fusions between antibodychains and other molecules, and the like.

The term “conventional molecular biology methods” refers to techniquesfor manipulating polynucleotides that are well known to the person ofordinary skill in the art of molecular biology. Examples of such wellknown techniques can be found in Molecular Cloning: A Laboratory Manual2nd Edition, Sambrook et al, Cold Spring Harbor, N.Y. (1989). Examplesof conventional molecular biology techniques include, but are notlimited to, in vitro ligation, restriction endonuclease digestion, PCR,cellular transformation, hybridization, electrophoresis, DNA sequencing,cell culture, and the like.

The term “variable region” as used herein in reference to immunoglobulinmolecules has the ordinary meaning given to the term by the person ofordinary skill in the act of immunology. Both antibody heavy chains andantibody light chains may be divided into a “variable region” and a“constant region”. The point of division between a variable region and aheavy region may readily be determined by the person of ordinary skillin the art by reference to standard texts describing antibody structure,e.g., Kabat et al “Sequences of Proteins of Immunological Interest: 5thEdition” U.S. Department of Health and Human Services, U.S. GovernmentPrinting Office (1991).

The present invention provides humanized antibody molecules specific forLK26 antigen in which at least parts of the CDRs of the heavy and/orlight chain variable regions of a human antibody (the receptor antibody)have been substituted by analogous parts of CDRs of a murine monoclonalantibody and the humanized antibody can specifically bind to the same asthe LK26 antibody. In a preferred embodiment of the subject invention,the CDR regions of the humanized LK26 specific antibody are derived fromthe murine antibody LK26. Some of the the humanized antibodies describedherein contain some alterations of the acceptor antibody, i.e., human,heavy and/or light chain variable domain framework regions that arenecessary for retaining binding specificity of the donor monoclonalantibody. In other words, the framework region of some embodiments thehumanized antibodies described herein does not necessarily consist ofthe precise amino acid sequence of the framework region of a naturaloccurring human antibody variable region, but contains varioussubstitutions that improve the binding properties of a humanizedantibody region that is specific for the same target as the murine LK26specific antibody. A minimal number of substitutions are made to theframework region in order to avoid large-scale introductions ofnon-human framework residues and to ensure minimal immunogenicity of thehumanized antibody in humans. The donor monoclonal antibody of thepresent invention is the LK26 murine antibody, which is specific for thehuman LK26 cancer antigen.

The humanized antibodies of the present invention include completeantibody molecules having full length heavy and light chains, or anyfragment thereof, such as the Fab or (Fab′)₂ fragments, a heavy chainand light chain dimer, or any minimal fragment thereof such as a Fv, anSCA (single chain antibody), and the like, specific for the LK26 antigenmolecule.

In addition to providing for humanized LK26 specific antibodies, thesubject invention provides for polynucleotides encoding humanized LK26specific antibodies. The subject polynucleotides may have a wide varietyof sequences because of the degeneracy of the genetic code. A person ofordinary skill in the art may readily change a given polynucleotidesequence encoding a humanized LK26 specific antibody into a differentpolynucleotide encoding the same humanized LK26 specific antibodyembodiment. The polynucleotide sequence encoding the antibody may bevaried to take into account factors affecting expression such as codonfrequency, RNA secondary structure, and the like.

The humanized antibodies of the subject invention may be produced by avariety of methods useful for the production of polypeptides, e.g. invitro synthesis, recombinant DNA production, and the like. Preferably,the humanized antibodies are produced by recombinant DNA technology.

The humanized LK26 specific antibodies of the invention may be producedusing recombinant immunoglobulin expression technology. The recombinantproduction of immunoglobulin molecules, including humanized antibodiesare described in U.S. Pat. No. 4,816,397 (Boss et al), U.S. Pat. No.4,816,567 (Cabilly et al) U.K. patent GB 2,188,638 (Winter et al), andU.K. patent GB 2,209,757. Techniques for the recombinant expression ofimmunoglobulins, including humanized immunoglobulins, can also be found,among other places in Goeddel et al, Gene Expression Technology Methodsin Enzymology Vol. 185 Academic Press (1991), and Borreback, AntibodyEngineering, W. H. Freeman (1992). Additional information concerning thegeneration, design, and expression of recombinant antibodies can befound in Mayforth, Desigining Antibodies, Academic Press, San Diego(1993).

The recombinant humanized anti-LK26 antibodies of the invention may beproduced by the following process or other recombinant proteinexpression methods:

a. Constructing, by conventional molecular biology methods, anexpression vector comprising an operon that encodes an antibody heavychain in which the CDRs and a minimal portion of the variable regionframework that are required to retain donor antibody binding specificityare derived from a non-human immunoglobulin, such as the murine LK26monoclonal antibody, and the remainder of the antibody is derived from ahuman immunoglobulin, thereby producing a vector for the expression of ahumanized antibody heavy chain.

b. Constructing, by conventional molecular biology methods, anexpression vector comprising an operon that encodes an antibody lightchain in which the CDRs and a minimal portion of the variable regionframework that are required to retain donor antibody binding specificityare derived from a non-human immunoglobulin, such as the murine LK26monoclonal antibody, and the remainder of the antibody is derived from ahuman immunoglobulin, thereby producing a vector for the expression ofhumanized antibody light chain.

c. Transferring the expression vectors to a host cell by conventionalmolecular biology methods to produce a transfected host cell for theexpression of humanized anti-LK26 antibodies.

d. Culturing the transfected cell by conventional cell culturetechniques so as to produce humanized anti-LK26 antibodies.

Host cells may be cotransfected with two expression vectors of theinvention, the first vector containing an operon encoding a heavy chainderived polypeptide and the second containing an operon encoding a lightchain derived polypeptide. The two vectors may contain differentselectable markers but, with the exception of the heavy and light chaincoding sequences, are preferably identical. This procedure provides forequal expression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes-both heavy and light chainpolypeptides. The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA or both.

The host cell used to express the recombinant antibody of the inventionmay be either a bacterial cell such as Escherichia coli, or preferably aeukaryotic cell. Preferably a mammalian cell such as a chinese hamsterovary cell, may be used. The choice of expression vector is dependentupon the choice of host cell, and may be selected so as to have thedesired expression and regulatory characteristics in the selected hostcell.

The general methods for construction of the vector of the invention,transfection of cells to produce the host cell of the invention, cultureof cells to produce the antibody of the invention are all conventionalmolecular biology methods. Likewise, once produced, the recombinantantibodies of the invention may be purified by standard procedures ofthe art, including cross-flow filtration, ammonium sulphateprecipitation, affinity column chromatography, gel electrophoresis andthe like.

The humanized LK26 specific antibodies of the present invention may beused in conjunction with, or attached to other antibodies (or partsthereof) such as human or humanized monoclonal antibodies. These otherantibodies may be reactive with other markers (epitopes) characteristicfor the disease against which the antibodies of the invention aredirected or may have different specificities chosen, for example, torecruit molecules or cells of the human immune system to the diseasedcells. The antibodies of the invention (or parts thereof) may beadministered with such antibodies (or parts thereof) as separatelyadministered compositions or as a single composition with the two agentslinked by conventional chemical or by molecular biological methods.Additionally the diagnostic and therapeutic value of the antibodies ofthe invention may be augmented by labelling the humanized antibodieswith labels that produce a detectable signal (either in vitro or invivo) or with a label having a therapeutic property. Some labels, e.g.radionucleotides may produce a detectable signal and have a therapeuticproperty. Examples of radionuclide labels include ¹²⁵I, ¹³¹I, ¹⁴C.Examples of other detectable labels include a fluorescent chromophoresuch as fluorescein, phycobiliprotein or tetraethyl rhodamine forfluorescence microscopy, an enzyme which produces a fluorescent orcolored product for detection by fluorescence, absorbance, visible coloror agglutination, which produces an electron dense product fordemonstration by electron microscopy; or an electron dense molecule suchas ferritin, peroxidase or gold beads for direct or indirect electronmicroscopic visualization. Labels having therapeutic properties includedrugs for the treatment of cancer, such as methotrexate and the like.

The subject invention also provides for a variety of methods fortreating and/or detecting cancer cells. These methods involve theadministration to of humanized LK26 specific antibodies, either labelledor unlabelled, to a patient. One method of detecting cancer cells in ahuman involves the step of administering a labeled humanized LK26specific antibody (labelled with a detectable label) to a human andsubsequently detecting bound labeled antibody by the presence of thelabel.

The recombinant antibodies of this invention may also be used for theselection and/or isolation of human monoclonal antibodies, and thedesign and synthesis of peptide or non-peptide compounds (mimetics)which would be useful for the same diagnostic and therapeuticapplications as the antibodies (e.g. Saragovi et al., (1991) Science253:792-795).

When the humanized LK26 specific antibodies of the invention are used invitro, the antibodies are typically administered in a compositioncomprising a pharmaceutical carrier. A pharmaceutical carrier can be anycompatible, non-toxic substance suitable for delivery of the monoclonalantibodies to the patient, Sterile water, alcohol, fats, waxes, andinert solids may be included in the carrier. Pharmaceutically acceptedadjuvants (buffering agents, dispersing agent) may also be incorporatedinto the pharmaceutical composition.

The humanized antibodies compositions of the invention may beadministered to a patient in a variety of ways. Preferably, thepharmaceutical compositions may be administered parenterally, i.e.,subcutaneously, intramuscularly or intravenously. Thus, this inventionprovides compositions for parenteral administration which comprise asolution of the human monoclonal antibody or a cocktail thereofdissolved in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., water, buffered water,0.4% saline, 0.3% glycine and the like. These solutions are sterile andgenerally free of particulate matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate, etc. The concentration of antibody inthese formulations can vary widely, e.g., from less than about 0.5%,usually at or at least about 1% to as much as 15 or 20% by weight andwill be selected primarily based on fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

Actual methods for preparing parenterally administrable compositions andadjustments necessary for administration to subjects will be known orapparent to those skilled in the art and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th Ed., MackPublishing Company, Easton, Pa. (1980), which is incorporated herein byreference.

The subject invention provide numerous humanized antibodies specific forthe LK26 antigen based on the discovery that the CDR regions of themurine monoclonal antibody could be spliced into a human acceptorframework so as to produce a humanized recombinant antibody specific forthe LK26 antigen. Preferred humanized LK26 specific antibodies containadditional change in the framework region (or in other regions) toincreasing binding for LK26 antigen. Particularly preferred embodimentsof the invention are the exemplified humanized antibody molecules havingsuperior binding properties for LK26.

The following examples are offered by way of illustration of theinvention, and should not be interpreted as a limitation of theinvention.

5.1. EXAMPLES

In the following examples all necessary restriction and modificationenzymes, plasmids and other reagents and materials were obtained fromcommercial sources unless otherwise indicated.

Unless otherwise indicated, all general recombinant DNA methodology wasperformed as described in “Molecular Cloning, A Laboratory Manual”(1989) Eds J. Sambrook et al., published by Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

In the following examples these abbreviations may be employed:

dCTP deoxycytidine triphosphate dATP deoxyadenosine triphosphate dGTPdeoxyguanosine triphosphate dTTP deoxythymidine triphosphate DTTdithiothreitol C cytosine A adenine G guanine T thymine PBS phosphatebuffered saline PBSB phosphate buffered saline containing 0.5% (w/v)bovine serum albumin PBST phosphate buffered saline containing 0.05%(v/v) Tween-20

5.1.1. Example 1 Production of Humanized Antibodies Specific for theLK26 Antigen

The source of the donor CDRs used to prepare these recombinantantibodies was a murine monoclonal antibody, mAbLK26, which is specificfor the LK26 antigen of certain human cancers. The LK26 monoclonalantibody was produced by immunization of (BALB/c x C57BL/6) F₁ mice withLU-75(c) choriocarcinoma cell lines and subsequent production andscreening of hybridoma cells. Cytoplasmic RNA was prepared from the mAbLK26 hybridoma cell line by the method of Favoloro, J. et al., (1980),Methods in Enzymology 65:718-749). cDNA was synthesized using Igvariable region primers as follows: for the Ig heavy chain variableregion (VH), the primer CG2aFOR (5′ GGAAGCTTAGACCGATGGGGCTGTTGTTTTG 3′)(SEQ ID NO: 1); for the light chain variable region (VK), the primerCK2FOR (5′ GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3′) (SEQ ID NO: 2). CDNAsynthesis reactions consisted of 5 μg RNA, 20 pmol CG2aFOR or CK2FOR,250 μM each of dATP, dCTP, dGTP and dTTP, 100 mM TrisHCl pH8.3, 140 mMKCl, 10 mM DTT, 10 mM MgCl₂ and 31.5 units of RNase inhibitor(Pharmacia, Milton Keynes, U.K.) in a total volume of 50 μl. Sampleswere heated to 70° C. for 10 minutes (min) then slowly cooled to 42° C.over a period of 30 min. 100 units of Moloney Murine Leukaemia virus(M-MLV) reverse transcriptase (Life Technologies Ltd, Paisley, U.K.) wasadded and incubation at 42° C. continued for 1 hour.

VH and VK cDNAs were then amplified using the polymerase chain reaction(PCR) as described by Saiki, R. K. et al., (1988), Science 239:487-491.The primers used were:

CG2aFOR (5′ GGAAGCTTAGACCGATGGGGCTGTTGTTTTG 3′) (SEQ ID NO: 1)

CK2FOR (5′ GGAAGCTTGAAGATGGATACAGTTGGTGCAGC 3′) (SEQ ID NO: 2)

VH1BACK (5′ AGGTSMARCTGCAGSAGTCWGG 3′) (SEQ ID NO: 3)

VK4BACK (5′ GACATTGAGCTCACCCAGTCTCCA 3′) (SEQ ID NO: 4)

where M=C or A, S=C or G, R=A or G and W=A or T. Such primers and theiruse in the PCR amplification of mouse Ig DNA are described by Orlandi,R. et al., (1989), Proc. Natl Acad. Sci. USA, 86:3833-3837. For PCRamplification of VH, 5 μl RNA/cDNA hybrid was mixed with 25 pmol CG2aFORand VH1BACK primers. For PCR amplification of VK, 5 μl RNA/cDNA hybridwas mixed with 25 pmol CK2FOR and VK4BACK primers. To these mixtures wasadded 200 μM each of DATP, dCTP, dGTP and dTTP, 67 mM TrisHCl pH8.8, 17mM (NH₄)₂SO₄, 10 mM MgCl₂, 0.02% (w/v) gelatin and 2.5 units of AmpliTaqDNA polymerase (Perkin Elmer Ltd, Beaconsfield, U.K.) in a total volumeof 50 μl. These were then subjected to 25 thermal cycles of PCR at 94°C., 30s; 50° C., 40s; 72° C., 30s; ending with 5 min at 72° C. Forcloning and sequencing, amplified DNA was purified by electrophoresis ina low melting point agarose gel and by Elutip-d column chromatography(Schleicher and Schuell, Dussel, Germany). Amplified VH DNA was cut withHindIII and PstI and cloned into M13mp18 or M13mp19 cut with HindIII andPstI (Life Technologies Ltd, Paisley, U.K.). Amplified VK DNA was cutwith HindIII and SacI and cloned into HindIII and SacI cut M13mp18 orM13mp19 (Life Technologies Ltd, Paisley, U.K,).

The resulting clones were sequenced by the dideoxy method (Sanger, F. etal., (1977), Proc. Natl Acad. Sci. USA 74:5463-5467) using Sequenase(United States Biochemical, Cleveland, Ohio, USA). The DNA and proteinsequences of the LK26 VH and VK domains are shown in FIGS. 1 and 2. Thelocation of the CDRs was determined with reference to Kabat, E. A. etal., (1987) “Sequences of Protein of Immunological Interest”, USDepartment of Health and Human Services, US Government Printing Office,and utilizing computer assisted alignment with other VH and VKsequences.

The transfer of the murine CDRs to human frameworks was achieved byoligonucleotide site-directed mutagenesis, based on the method ofNakamye, K. and Eckstein, F. (1986) Nucleic Acids Res. 14:9679-9698. Thehuman framework regions chosen to receive the transplanted CDRs wereNEWM or KOL and REI for the heavy and light chains respectively. Thestructures of these proteins have been solved crystallographically. Thetemplates for mutagenesis were human framework region genes containingirrelevant CDRs and consisted of synthetic DNAs cloned into M13 phage(Riechmann, L. et al., (1988) Nature 332:323-327).

The oligonucleotides used were:

NEWH VH: VHCDR1 5′TGGCTGTCTCACCCAAGACAAGCCATAGCCGCTGAAGGTG (SEQ ID NO:5)  AAGCCAGACGCGGTGCAGGTCAGGCT 3′ VHCDR25′GTTCTTGCTGGTGTCTCTCAGCATTGTCACTCTCCCCTTCAC (SEQ ID NO:6)ACTGTCTGCATAGTAGGTATAACTACCACCACTACTAATCATTG CAACCCACTCAAGACC 3′ VHCDR35′TGAGGAGACGGTGACCAGGCTCCCTTGGCCCCAGTAAGCAA (SEQ ID NO:7)ACCAGGCGGGATCGTCCCCATGTCTTGCACAATAATA 3′ KOL VH: VHCDR15′CCTGTCTCACCCAAGACAACCCATAGCCGCTGAAGGTGAAGC (SEQ ID NO:8)CAGATGCGGAGCAGGACAGGC 3′ VHCDR25′GAACAATGTGTTCTTGGCGTTGTCTCGCGATATTGCAAATCT (SEQ ID NO:9)ACCCTTCACACTGTCTGCATAGTAGGTATAACTACCACCACTACTAATCATTGCAACCCACTCAAGACCTTTTCC 3′ VHCDR35′CCAATAAGCAAACCAGGCGGGATCGTCCCCATGTCTTGCA (SEQ ID NO:10) CAAAAATAGAC 3′REI VK: VKCDR1 5′ CTTCTGCTGGTACCAGTGCAAGTTGTTGGAACTTATACTT (SEQ IDNO:11) GAGCTGACACTACAGGTGATGGTCAC 3′ VKCDR2 5′TCTGCTTGGCACACCAGAAGCCAGGTTGGATGTGCCGTA (SEQ ID NO:12) GATCAGCAGCTT 3′VKCDR3 5′ GGTCCCTTGGCCGAACGTGTACATGTACGGGTAACTACT (SEQ ID NO:13)CCACTGTTGGCAGTAGTAGGTGGC 3′

A number of additional, murine residues were introduced into thevariable region frameworks by extension of the CDR primers.Specifically:

NEWM V(24) changed to A (NEWM VHCDR1 oligonucleotide) NEWM S(27) changedto F (NEWM VHCDR1 oligonucleotide) NEWM I(48) changed to V (NEWM VHCDR2oligonucleotide) NEWM G(49) changed to A (NEWM VHCDR2 oligonucleotide)NEWM V(71) changed to R (NEWM VHCDR2 oligonucleotide) KOL S(24) changedto A (KOL VHCDR1 oligonucleotide) KOL I(28) changed to T (KOL VHCDR1oligonucleotide) KOL T(68) changed to A (KOL VHCDR2 oligonucleotide) KOLS(74) changed to A (KOL VHCDR2 oligonucleotide)

These residues that have been changed are believed to be important forretaining original antigen specificity. Although the invention is notdependent upon any particular explanation for the results obtained bymaking the additional residue changes, some possible explanations fortheir significance are as follows:

The change of residues NEWM V(24) and KOL S(24) to the smaller Afacilitates the accommodation of the heterologous CDR1 loop. The NEWMS(27) to F change was made because S(27) is an unusual residue insubgroup II human heavy chains (Riechmann et al., (1988) Nature332:323-327). Amino acids VH(27-30, 47-49, 71) are residues of the‘vernier zones’ as defined by Foote and Winter (Foote, J. and Winter G.(1992) J. Mol. Biol. 224:487-499. These zones are important foradjusting CDR structures to promote antigen binding. This explanationaccounts for the changes NEWM S(27) to F, NEWM I(48) to V, NEWM G(49) toA, NEWM V(71) to R and KOL I(28) to T.

For site directed mutagenesis the VH and VK oligonucleotides encodingthe murine CDRs were phosphorylated with T4 Kinase (Life TechnologiesLtd, Paisley, U.K.). A 25 fold molar excess of each of the three VH orVK primers were added to 0.5 μg of appropriate VH or VK single strandedtemplate DNA in M13 (NEWM VH : M13VHPCR1; KOL VH M13MN14VH; REI :M13VKPCR2) in 40 mM Tris HCl pH7.5, 20 mM MgCl₂, 50 mM NaCl and annealedby heating to 90° C. for a few minutes and slowly cooling to 37° C. Theannealed DNA was extended with 2.5 units of T7 DNA polymerase (cloned,United States Biochemical, Cleveland, Ohio, USA) in a reaction mixturecontaining 0.5 units of T4 DNA ligase (Life Technologies Ltd, Paisley,U.K.), 0.25 mM of each of dATP, dGTP, dTTP, and dCTP (Pharmacia, MiltonKeynes, U.K.), 40 mM Tris HCl pH7.5, 20 mM MgCl₂, 50 mM NaCl, 6.5 mM DTTand 1 mM ATP in a total volume of 30 μl. The mixture was incubated atroom temperature for 1 h. A 1 μl aliquot of this extension/ligationmixture was then used in an asymmetric PCR for the specificamplification of the newly synthesized strand. The reaction contained 1μl extension/ligation mixture, 250 μM of each of dATP, dGTP, dTTP anddCTP, 67 mM Tris HCl pH8.8, 17 mM (NH₄)₂SO₄, 10 mM MgCl₂, 0.02% (w/v)gelatin, 2.5 units of AmpliTaq DNA polymerase and 25 pmol of appropriateoligonucleotide primer (5′ AACAGCTATGACCATG 3′ (SEQ ID NO: 14) for NEWMVH and KOL VH; 5′ CTCTCTCAGGGCCAGGCGGTGA 3′ (SEQ ID NO: 15) for REI VK)in a total volume of 50 μl. The reaction mixtures were subjected to 30thermal cycles of PCR at 94° C., 30 s; 55° C., 30 s; 72° C., 1 minending with 72° C., 5 min. The newly synthesized strand was thenamplified by adding 20 pmol of appropriate oligonucleotide primer (5′GTAAAACGACGGCCAGT 3′ (SEQ ID NO: 16) for NEWM VH and KOL VH and 5′GCGGGCCTCTTCGCTATTACGC 3′ (SEQ ID NO: 17) for REI VK) and adjusting thereaction mixture to include a further 5 n moles of each of dATP, dGTP,dTTP and dCTP and 2.5 Units of AmpliTaq. The reactions were subjected toa further 20 PCR cycles as above. The amplified VH and VK DNAs werepurified from 1.5% w/v low melting point agarose gels by elutip-d columnchromatography. Purified DNA was digested with HindIII and BamHI plusRsaI (for VHs) or BstXI (for VKs) (all restriction enzymes were obtainedfrom Life Technologies Ltd, Paisley, U.K.). There is an RsaI site in theparental VHPCR1 and MN14VH and a BstXI site in the parental VKPCR2 butthese sites are deleted during mutagenesis. These digestions thereforeselect for newly synthesized DNA. The HindIII/BamHI digested VH and VKDNAs were ligated into HindIII/BamHI cut M13mp18 or M13mp19 (both fromPharmacia, Milton Keynes, U.K.) and transformed into competent E. coliTG1 (Amersham International plc, Amersham, U.K.). Single stranded DNAwas prepared from individual ‘plaques’ and sequenced by the dideoxymethod using Sequenase (United States Biochemical, Cleveland, Ohio, USA)according to Manufacturer's instructions. Triple CDR mutants wereidentified in this way and selected for construction of VH and VKexpression vectors.

The expression vectors for the humanized VH and VK genes, pSVgpt andpSVhyg are shown in FIGS. 3 and 4. The humanized VH genes, together withthe immunoglobulin heavy chain promoter, appropriate splice sites andsignal peptide sequences were excised from the M13 clones with HindIIIand BamHI and ligated into the heavy chain expression vector, pSVqpt.This vector contains the murine heavy chain immunoglobulin enhancer, theqpt gene under control of the SV40 promoter/enhancer for selection inmammalian cells, the human IgG1 constant region domain and sequences forreplication and selection in E. coli. The humanized VK gene was clonedinto the light chain expression vector pSVhyg in the same way. Allfeatures of pSVhyg are the same as in pSVgpt except that the qpt gene isreplaced by the gene for hygromycin resistance (hyg) and a human kappaconstant region is included instead of the IgG1 constant region.

For transfection into mammalian cells 10 μg of the heavy chainexpression vector DNA and 20 μg of the light chain vector DNA werelinearized by digestion with PvuI (Life Technologies Ltd, Paisley,U.K.), coprecipitated with ethanol and redissolved in 20 μg of water.The recipient cell line was NSO, a non-immunoglobulin producing mousemyeloma, obtained from the European collection of Animal Cell Cultures,Porton, U.K., ECAC No. 85110505 cells were grown in Dulbecco's ModifiedEagle's Medium supplemented with 10% foetal calf serum and antibiotics(DMEM) (Life Technologies Ltd, Paisley, U.K.). Approximately 10⁷ NSOcells were harvested by centrifugation and resuspended in 0.5 ml DMEM,the digested DNA was added and the cells transferred to a cuvette andplaced on ice for 5 min. A single pulse of 170 volts, 960 μ farads wasadministered (Genepulser, BioRad, Richmond, Calif., U.S.A.). After afurther 30 min on ice the cells were replaced in a flask in 20 ml DMEMand allowed to recover for 24 hours. After this time the cells weredistributed into a 24 well plate in selective medium (DMEM with 0.8μg/ml mycophenolic acid and 250 μg/ml xanthine). After 3 to 4 days themedium was changed for fresh selective medium. Colonies of transfectedcells were visible after 10 to 14 days.

The production of human antibody in the wells containing transfectedclones was measured by ELISA. Capture antibody, goat anti-human IgG,gamma chain specific (Sera-Lab Ltd, Crawley Down, U.K.) was diluted to 5μg/ml in 50 mM carbonate buffer pH9.6, and used to coat polystyreneELISA plates (Dynatech Immulon 1), 200 μl per well, overnight at 4° C.After washing 3 times with PBST, 50-100 μl of the culture medium to bescreened was added to the wells and incubated at 37° C. for 60 min. Thewells were washed again with PBST and the reporter antibody,peroxidase-conjugated goat anti-human IgG, gamma chain specific(Sera-Lab Ltd, Crawley Down, U.K.) or peroxidase-conjugated goatanti-human kappa chain (Sera-Lab Ltd, Crawley Down, U.K) was added at100 ng per well and the plate incubated for a further 60 min. The platewas washed as before then the colour was developed. Substrate buffer wasprepared by mixing 100 mM citric acid and 100 mM disodium hydrogenphosphate to pH5.0. 25 mg of o-phenylenediamine was dissolved in 50 mland 5 μl of 30% hydrogen peroxide added just before use. 200 μl wasdispensed per well and incubated at room temperature in the dark. Thereaction was stopped by addition of 50 μl per well of 12.5% sulphuricacid and the absorbances were read at 492 nm.

Positive cell clones were expanded for antibody purification. For thefinal expansion to production volume the cells were diluted in DMEMcontaining 10% IgG-free fetal calf serum. For small scale purification500 ml of conditioned medium from static flask or spinner cultures washarvested by centrifugation. 0.1 volumes of 1.0M TrisHCl pH8.0 and 0.5to 1.0 ml of Protein A-agarose (Boehringer Mannheim, Lewes, U.K.) wereadded. This was stirred overnight at room temperature then collected ona disposable column. This was washed with 10 column volumes of 0.1MTrisHCl pH8.0, 10 column volumes of 0.01 M TrisHCl pH8.0 and eluted with0.1M glycine buffer, pH3.0. 1.0 ml fractions were collected into tubescontaining 100 μl of 1.0M TrisHCl , pH8.0. Fractions containing antibodywere pooled and dialysed against PBS. The concentrations of the antibodypreparations were determined using a Micro BCA Protein Assay Reagent Kit(Pierce, Rockford, USA). Samples were checked by running on 10%SDS-polyacrylamide gels.

The chimeric LK26 antibody, in which the murine constant region domainsof the heavy and light chains had been replaced by the human constantregions used in the humanized antibody, was constructed as described byOrlandi et al., (1989). Three hybrid chimeric/humanized antibodies wereconstructed consisting of the chimeric heavy chain with the humanizedlight chain and the humanized heavy chain (from NEWM and KOLmutagenesis) with the chimeric light chain.

None of these hybrid antibodies showed binding to the SW620 target cellsequivalent to the chimeric antibody. This indicates that furtherframework changes, in both the VH and VK chains, are necessary torestore antigen binding.

Four further versions of the LK26HuVH and two further versions of theLK26HuVK were designed. The amino acid sequences of these VHs and VKsare shown in Table 1.

Table 1 shows the variable region sequences of LK26HuVH,LK26HuVHFAIS,N(SEQ ID NO: 19), LK26HuVH SLF(SEQ ID NO: 21),LK26HuVHI,I(SEQ ID NO: 20), LK26KOLHuVH (SEQ ID NO: 22), LK26HuVK (SEQID NO: 23), LK26HuVKY (SEQ ID NO: 24) and LK26HuVKPW,Y (SEQ ID NO: 25with the proviso that amino acid 72 is not phe but is Tyr). Murineframework residues are shown in lower case. Some framework residues inNEWM and REI are unusual for human subgroup II heavy chains or humansubgroup I kappa chains, respectively, these have been replaced by theresidues commonly found at these positions and are underlined in thetable.

The additional changes to the HuVH and HuVK constructs are shown below(numbering according to Kabat et al., ibid):

LK26HuVHFAIS,N (68-71, 74)

LK26HuVHSLF (78-80)

LK26HuVHI,I (93-95)

LK26HuVKY (72).

LK26HuVKPW,Y (47-48, 72)

These new versions were constructed by mutagenesis of the originalreshaped heavy and light chain M13 single stranded DNA clones. Themethod of Higuchi, R. et al. (1988) Nucleic Acids Res. 16:7351-7367,which utilizes overlapping PCR amplification with mutagenic primers, wasemployed. The modified variable regions were cloned into the expressionvector pSVgpt or pSVhyg as before and cotransfected with either the MuVKor MuVH plasmids into NSO cells. Antibody producing cell clones wereselected, expanded and purified for testing. Subsequent to this, fullyhumanized version antibodies consisting of the modified HuVHs and HuVKswere prepare in the same way.

Another version of the humanized light chain produced was LK26HuVKPW(SEQ ID NO: 25). LK26HuVKPW (SEQ ID NO: 25) is similar to LK26HuVKPW,Y(SEQ ID NO: 25, with the proviso that amino acid 72 is not Phe but isTyr), but lacks the change at position 71.

5.1.2. Example 2 Specific Binding of Humanized LK26 Antibodies toCarcinoma Cells

The recombinant antibodies have been tested in ELISAs using the SW620target cells. The ELISA method used is as follows:

SW620 cells are diluted to 1.5×10⁵−2.5×10⁵ cells/ml in DMEM, 10% FCS and20 μl (ie 3-5×10⁴ cells) added to each well. Cells are grown untilnearly confluent (about 2 days). Plates are washed 2× with PBS and 100μl antibody (diluted in DMEM) added. Incubation is carried out at 4° C.for 1 hour. The wells are washed 3× with PBS and 100 μl of appropriatereporter antibody added, ie either goat anti-human IgG1, HRPO conjugate(Sera-lab, 0.4 mg/ml, diluted 1: 500 in DMEM) or goat anti-mouse IgG1,HRPO conjugate (Sera-lab, 0.4 mg/ml, diluted 1:500 in DMEM), incubationis carried out at 4° C. for 1 hour. Wells are washed 3× with PBS andbound reporter antibody detected using H₂O₂ ando-phenylenediaminedihydrochloride and the OD 492 nm measured.

The humanized antibodies tested in ELISAs are LK26HuVHSLF (SEQ ID NO:21)/HuVKPW,Y (SEQ ID NO: 25 with the proviso that amino acid 72 is notPhe but is Tyr), LK26HuVHFAIS,N (SEQ ID NO: 19)/HuVKPW,Y (SEQ ID NO: 25with the proviso that amino acid 72 is not Phe but is Tyr) andLK-26KOLHUVH/HuVKPW,Y. The data are presented graphically below. Thetest data of the various recombinant antibodies indicate that ofparticular value for the restoration of antigen binding is the inclusionof the P and W residues into the humanized light chain. Furthermore, thedata show that these inclusions facilitate the proper association of theVH and VL domains. This invention therefore also relates to theinclusion into the humanized antibody of these and other VH and VLresidues to facilitate proper VH and VL association and thereby antigenbinding.

The test data indicates that these humanized antibodies retain thebinding properties of the original murine and chimeric antibodies. Inparticular the LK26HuVHFAIS,N/HuVKPW,Y (SEQ ID NO: 25 with the provisothat amino acid 72 is not Phe but is Tyr) and the LK26KOLHuVH (SEQ IDNO: 22)/HuVKPW,Y (SEQ ID NO: 25 with the proviso that amino acid is notPhe but is Tyr) exhibit binding affinities higher than the chimericantibody. Such recombinant antibodies (of which these are examples)therefore provide for novel, recombinant antibody molecules for thediagnosis and therapy of human cancers characterized by the expressionof the LK26 antigen.

TABLE 1 LK26HuVH: QVQLQESGPGLVRPSQTLSLTCTaSGfTFSGYGLSWVRQ (SEQ ID NO:18)PPGRGLEWvaMISSGGSYTYYADSVKGRVTMLrDTSKNQFSLRLSSVTAADTAVYYCARHGDDPAWFAYWGQGSLVTV SS LK26HuVHFAIS,N:QVQLQESGPGLVRPSQTLSLTCTaSGfTFSGYGL (SEQ ID NO:19)SWVRQPPGRGLEWvaMISSGGSYTYYADSVKGRf aisrDnSKNQFSLRLSSVTAADTAVYYCARHGDDPAWFAYWGQGSLVTVSS LK26HuVHI,I: QVQLQESGPGLVRPSQTLSLTCTaSGfTFSGYGLSWVRQ(SEQ ID NO:20) PPGRGLEWvaMISSGGSYTYYADSVKGRVTMLrDTSRNQFSLRLSSYTAADTAiYiCARHGDDPAWFAYWGQGSLVTV SS LK26HuVHSLF:QVQLQESGPGLVRPSQTLSLTCTaSGfTFSGYGLSWVRQ (SEQ ID NO:21)PPGRGLEWvaMISSGGSYTYYADSVKGRVTMLrDTSKNs1fLRLSSVTAADTAVYYCARHGDDPAWFAYWGQGTTVTV SS LK26KOLHuVH:EVQLVESGGGVVQPGRSLRLSCSaSGFtFSGYGLSWVRQ (SEQ ID NO:22)APGKGLEWVAMISSGGSYTYYADSVKGRFaISRDNaKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTV SS LK26HuVK:DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQ (SEQ ID NO:23)KPGKAPKLLIYGTSNLASGVPSRFSGSGSGTDFTFTISS LQPEDIATYYCQQWSSYPYMYTFGQGTKVEIKLK26HuVKY: DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQ (SEQ ID NO:24)KPGKAPKLLIYGTSNLASGVPSRFSGSGSGTDYTFTISS LQPEDIATYYCQQWSSYPYMYTFGQGTKVEIKLK26HuVKPW,Y: DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKpwIYGTSNLASGVPSRFSGSGSGTDFTFTISS LQPEDIATYYCQQWSSYPYMYTFGQGTKVEIK(SEQ ID NO:5) with the proviso that amino acid 72 is not Phe but is Tyr)

Incorporation by Reference

All patents, patents applications, and publications cited areincorporated herein by reference.

Equivalents

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Indeed, variousmodifications of the above-described makes for carrying out theinvention which are obvious to those skilled in the field of molecularbiology or related fields are intended to be within the scope of thefollowing claims.

31 31 base pairs nucleic acid unknown unknown DNA (genomic) 1 GGAAGCTTAGACCGATGGGG CTGTTGTTTT G 31 32 base pairs nucleic acid unknown unknownDNA (genomic) 2 GGAAGCTTGA AGATGGATAC AGTTGGTGCA GC 32 22 base pairsnucleic acid unknown unknown DNA (genomic) 3 AGGTSMARCT GCAGSAGTCW GG 2224 base pairs nucleic acid unknown unknown DNA (genomic) 4 GACATTGAGCTCACCCAGTC TCCA 24 66 base pairs nucleic acid unknown unknown DNA(genomic) 5 TGGCTGTCTC ACCCAAGACA AGCCATAGCC GCTGAAGGTG AAGCCAGACGCGGTGCAGGT 60 CAGGCT 66 102 base pairs nucleic acid unknown unknown DNA(genomic) 6 GTTCTTGCTG GTGTCTCTCA GCATTGTCAC TCTCCCCTTC ACACTGTCTGCATAGTAGGT 60 ATAACTACCA CCACTACTAA TCATTGCAAC CCACTCAAGA CC 102 78 basepairs nucleic acid unknown unknown DNA (genomic) 7 TGAGGAGACG GTGACCAGGCTCCCTTGGCC CCAGTAAGCA AACCAGGCGG GATCGTCCCC 60 ATGTCTTGCA CAATAATA 78 63base pairs nucleic acid unknown unknown DNA (genomic) 8 CCTGTCTCACCCAAGACAAC CCATAGCCGC TGAAGGTGAA GCCAGATGCG GAGCAGGACA 60 GGC 63 117base pairs nucleic acid unknown unknown DNA (genomic) 9 GAACAATGTGTTCTTGGCGT TGTCTCGCGA TATTGCAAAT CTACCCTTCA CACTGTCTGC 60 ATAGTAGGTATAACTACCAC CACTACTAAT CATTGCAACC CACTCAAGAC CTTTTCC 117 51 base pairsnucleic acid unknown unknown DNA (genomic) 10 CCAATAAGCA AACCAGGCGGGATCGTCCCC ATGTCTTGCA CAAAAATAGA C 51 66 base pairs nucleic acid unknownunknown DNA (genomic) 11 CTTCTGCTGG TACCAGTGCA AGTTGTTGGA ACTTATACTTGAGCTGACAC TACAGGTGAT 60 GGTCAC 66 51 base pairs nucleic acid unknownunknown DNA (genomic) 12 TCTGCTTGGC ACACCAGAAG CCAGGTTGGA TGTGCCGTAGATCAGCAGCT T 51 63 base pairs nucleic acid unknown unknown DNA (genomic)13 GGTCCCTTGG CCGAACGTGT ACATGTACGG GTAACTACTC CACTGTTGGC AGTAGTAGGT 60GGC 63 16 base pairs nucleic acid unknown unknown DNA (genomic) 14AACAGCTATG ACCATG 16 22 base pairs nucleic acid unknown unknown DNA(genomic) 15 CTCTCTCAGG GCCAGGCGGT GA 22 17 base pairs nucleic acidunknown unknown DNA (genomic) 16 GTAAAACGAC GGCCAGT 17 22 base pairsnucleic acid unknown unknown DNA (genomic) 17 GCGGGCCTCT TCGCTATTAC GC22 119 amino acids amino acid unknown unknown protein 18 Gln Val Gln LeuGln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu SerLeu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Leu SerTrp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Met IleSer Ser Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly ArgVal Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu ArgLeu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala ArgHis Gly Asp Asp Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 SerLeu Val Thr Val Ser Ser 115 119 amino acids amino acid unknown unknownprotein 19 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro SerGln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe SerGly Tyr 20 25 30 Gly Leu Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu GluTrp Val 35 40 45 Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ala AspSer Val 50 55 60 Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn GlnPhe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala ValTyr Tyr Cys 85 90 95 Ala Arg His Gly Asp Asp Pro Ala Trp Phe Ala Tyr TrpGly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser 115 119 amino acidsamino acid unknown unknown protein 20 Gln Val Gln Leu Gln Glu Ser GlyPro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys ThrAla Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Leu Ser Trp Val Arg GlnPro Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Met Ile Ser Ser Gly GlySer Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Val Thr Met LeuArg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser ValThr Ala Ala Asp Thr Ala Ile Tyr Ile Cys 85 90 95 Ala Arg His Gly Asp AspPro Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr ValSer Ser 115 119 amino acids amino acid unknown unknown protein 21 GlnVal Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30Gly Leu Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Val 35 40 45Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Ser Leu Phe 65 70 7580 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 9095 Ala Arg His Gly Asp Asp Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100105 110 Thr Thr Val Thr Val Ser Ser 115 119 amino acids amino acidunknown unknown protein 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly ValVal Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser GlyPhe Thr Phe Ser Gly Tyr 20 25 30 Gly Leu Ser Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Val 35 40 45 Ala Met Ile Ser Ser Gly Gly Ser Tyr ThrTyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ala Ile Ser Arg Asp AsnAla Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro GluAsp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Arg His Gly Asp Asp Pro Ala TrpPhe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser 115110 amino acids amino acid unknown unknown protein 23 Asp Ile Gln LeuThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg ValThr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Asn 20 25 30 Asn Leu HisTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr GlyThr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser GlySer Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro GluAsp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 Tyr MetTyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 110 aminoacids amino acid unknown unknown protein 24 Asp Ile Gln Leu Thr Gln SerPro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile ThrCys Ser Val Ser Ser Ser Ile Ser Ser Asn 20 25 30 Asn Leu His Trp Tyr GlnGln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Thr Ser AsnLeu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly ThrAsp Tyr Thr Phe Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Ile AlaThr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 Tyr Met Tyr Thr PheGly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 110 amino acids aminoacid unknown unknown protein 25 Asp Ile Gln Leu Thr Gln Ser Pro Ser SerLeu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser ValSer Ser Ser Ile Ser Ser Asn 20 25 30 Asn Leu His Trp Tyr Gln Gln Lys ProGly Lys Ala Pro Lys Pro Trp 35 40 45 Ile Tyr Gly Thr Ser Asn Leu Ala SerGly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe ThrPhe Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Ile Ala Thr Tyr TyrCys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 Tyr Met Tyr Thr Phe Gly Gln GlyThr Lys Val Glu Ile Lys 100 105 110 357 base pairs nucleic acid unknownunknown DNA (genomic) 26 CAGGTSMARC TGCAGSAGTC WGGGGGAGAC TTGGTGAAGCCTGGAGGGTC CCTGAAACTC 60 TCCTGTGCAG CCTCTGGATT CACTTTCAGT GGCTATGGCTTGTCTTGGGT TCGCCAGACT 120 CCAGACAAGA GGCTGGAGTG GGTCGCAATG ATTAGTAGTGGTGGTAGTTA TACCTACTAT 180 GCAGACAGTG TGAAGGGGCG ATTCGCCATC TCCAGAGACAATGCCAAGAA CTCCCTGTTC 240 CTGCAAATGA GCAGTCTGAA GTCTGACGAC ACAGCCATTTATATCTGTGC AAGACATGGG 300 GACGATCCCG CCTGGTTTGC TTACTGGGGC CAAGGGACTCTAGTCACTGT CTCTGCT 357 357 base pairs nucleic acid unknown unknown DNA(genomic) 27 AGCAGAGACA GTGACTAGAG TCCCTTGGCC CCAGTAAGCA AACCAGGCGGGATCGTCCCC 60 ATGTCTTGCA CAGATATAAA TGGCTGTGTC GTCAGACTTC AGACTGCTCATTTGCAGGAA 120 CAGGGAGTTC TTGGCATTGT CTCTGGAGAT GGCGAATCGC CCCTTCACACTGTCTGCATA 180 GTAGGTATAA CTACCACCAC TACTAATCAT TGCGACCCAC TCCAGCCTCTTGTCTGGAGT 240 CTGGCGAACC CAAGACAAGC CATAGCCACT GAAAGTGAAT CCAGAGGCTGCACAGGAGAG 300 TTTCAGGGAC CCTCCAGGCT TCACCAAGTC TCCCCCWGAC TSCTGCAGYTKSACCTG 357 119 amino acids amino acid unknown unknown protein 28 GlnVal Xaa Leu Gln Xaa Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30Gly Leu Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 7580 Leu Gln Met Ser Ser Leu Lys Ser Asp Asp Thr Ala Ile Tyr Ile Cys 85 9095 Ala Arg His Gly Asp Asp Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100105 110 Thr Leu Val Thr Val Ser Ala 115 330 base pairs nucleic acidunknown unknown DNA (genomic) CDS 1..330 29 GAC ATT GAG CTC ACC CAG TCTCCA GCA CTC ATG GCT GCA TCT CCA GGG 48 Asp Ile Glu Leu Thr Gln Ser ProAla Leu Met Ala Ala Ser Pro Gly 1 5 10 15 GAG AAG GTC ACC ATC ACC TGCAGT GTC AGC TCA AGT ATA AGT TCC AAC 96 Glu Lys Val Thr Ile Thr Cys SerVal Ser Ser Ser Ile Ser Ser Asn 20 25 30 AAC TTG CAC TGG TAC CAG CAG AAGTCA GAA ACC TCC CCC AAA CCC TGG 144 Asn Leu His Trp Tyr Gln Gln Lys SerGlu Thr Ser Pro Lys Pro Trp 35 40 45 ATT TAT GGC ACA TCC AAC CTG GCT TCTGGA GTC CCT CTT CGC TTC AGA 192 Ile Tyr Gly Thr Ser Asn Leu Ala Ser GlyVal Pro Leu Arg Phe Arg 50 55 60 GGC TTT GGA TCT GGG ACC TCT TAT TCT CTCACA ATC AGC AGC ATG GAG 240 Gly Phe Gly Ser Gly Thr Ser Tyr Ser Leu ThrIle Ser Ser Met Glu 65 70 75 80 GCT GAA GAT GCT GCC ACT TAT TAC TGT CAACAG TGG AGT AGT TAC CCG 288 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln GlnTrp Ser Ser Tyr Pro 85 90 95 TAC ATG TAC ACG TTC GGA GGG GGG ACC AAG TTGGAA ATA AAA 330 Tyr Met Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys100 105 110 110 amino acids amino acid unknown protein 30 Asp Ile GluLeu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly 1 5 10 15 Glu LysVal Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Asn 20 25 30 Asn LeuHis Trp Tyr Gln Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp 35 40 45 Ile TyrGly Thr Ser Asn Leu Ala Ser Gly Val Pro Leu Arg Phe Arg 50 55 60 Gly PheGly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 AlaGlu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 TyrMet Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 330 basepairs nucleic acid unknown unknown DNA (genomic) 31 TTTTATTTCCAACTTGGTCC CCCCTCCGAA CGTGTACATG TACGGGTAAC TACTCCACTG 60 TTGACAGTAATAAGTGGCAG CATCTTCAGC CTCCATGCTG CTGATTGTGA GAGAATAAGA 120 GGTCCCAGATCCAAAGCCTC TGAAGCGAAG AGGGACTCCA GAAGCCAGGT TGGATGTGCC 180 ATAAATCCAGGGTTTGGGGG AGGTTTCTGA CTTCTGCTGG TACCAGTGCA AGTTGTTGGA 240 ACTTATACTTGAGCTGACAC TGCAGGTGAT GGTGACCTTC TCCCCTGGAG ATGCAGCCAT 300 GAGTGCTGGAGACTGGGTGA GCTCAATGTC 330

What is claimed is:
 1. A method for treating a subject with a cancer, cells of which present antigen LK26 on their surfaces, comprising administering to said subject a therapeutically effective amount of a humanized antibody specific for said LK26 antigen, wherein said humanized antibody comprises a variable region heavy chain and a variable region light chain, wherein said variable region heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 22, and said variable region light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 25, with the proviso that, in SEQ ID NO: 25, amino acid 72 is Tyr, wherein said humanized antibody is conjugated to a label having a therapeutic property.
 2. The method of claim 1, wherein said label is a radionuclide.
 3. The method of claim 2, wherein said radionuclide is ¹²⁵I, ¹³¹I, or ¹⁴C.
 4. The method of claim 1, wherein said label is an anticancer drug.
 5. The method of claim 4, wherein said anticancer drug is methotrexate.
 6. The method of claim 1, wherein said variable region heavy chain comprises SEQ ID NO: 22, and said variable region light chain comprises SEQ ID NO:
 25. 7. The method of claim 1, wherein said variable region heavy chain comprises SEQ ID NO: 21, and said variable region light chain comprises SEQ ID NO:
 25. 8. The method of claim 1, wherein said variable region heavy chain comprises SEQ ID NO: 19, and said variable region light chain comprises SEQ ID NO:
 25. 9. The method of claim 1, wherein said cancer is choriocarcinnoma, teratocarcinoma, or renal cancer. 